Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-07-09
2001-04-17
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S091500, C435S173100, C536S023100, C536S024310, C536S024330, C536S025320
Reexamination Certificate
active
06218118
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods and reagents for analyzing nucleotide sequences of nucleic acids via mass spectrometry and, more particularly, to methods for analyzing nucleotide sequences employing reagents that are mixtures of oligonucleotide precursors having a high level of mass number complexity and sequence- coverage complexity.
BACKGROUND OF THE INVENTION
Determining the nucleotide sequence of nucleic acids (DNA and RNA) is critical to understanding the function and control of genes and their relationship, for example, to disease discovery and disease management. Analysis of genetic information plays a crucial role in the biological experimentation. This has become especially true with regard to studies directed at understanding the fundamental genetic and environmental factors associated with disease and the effects of potential therapeutic agents on the cell. This paradigm shift has lead to an increasing need within the life science industries for more sensitive, more accurate and higher-throughput technologies for performing analysis on genetic material obtained from a variety of biological sources.
Because sequencing the enormously large number of nucleic acids in each human cell is necessarily a time-consuming process, there is always a pressing need for faster and higher through-put analyses that do not sacrifice sensitivity and accuracy. A number of techniques have been developed, including, inter alia, electrophoresis, enzymatic and chemical analysis, array technology and mass spectrometry, to determine the nucleotide sequence of nucleic acids.
Electrophoretic Techniques
Slab or capillary polyacrylamide gel electrophoresis technologies, such as those employed in automated DNA sequencers, provide highly accurate de novo sequence information for relatively long (500-700 residues or bases) segments of DNA. Although electrophoresis-based techniques provide a great amount of information per sample, they require long sample preparation and set-up times and thereby limit throughput.
Enzymatic and Chemical Analysis
A number of enzymatic and chemical techniques exist to determine the de novo nucleotide sequence of nucleic acids. However, each technique has inherent limitations. For example, Maxam and Gilbert [Proc. Natl. Acad. Sci. USA 74:5460 (1977)] disclose a chemical degradation approach and Sanger et al. [Proc. Natl. Acad. Sci. USA 74:5463 (1977)] disclose a chain termination method using complementary strand primer extension. Each of these techniques utilizes four separate reaction mixtures to create a nested set of fragments differing by a single nucleotide in length, thus representing a complete nucleotide sequence. A resolution of the fragments based on their size and terminating nucleotide is carried out to determine the order of the fragments and hence the nucleotide sequence.
Single-stranded conformation polymorphism (SSCP) analysis a useful technique for detecting relatively small differences among similar sequences. The technique is simple to implement and, when combined with multiple-dye detection or mass-tag methodologies, may be multiplexed and thereby improve throughput. However, like techniques that rely on detecting heteroduplexes, such as denaturing gradient gel electrophoresis (DGGE), chemical cleavage (CCM), enzymatic cleavage (using cleavase) of mismatches, and denaturing high performance liquid chromatography (DHPLC), the technique is only qualitative, i.e., the technique only reveals whether a mutation is present within the target nucleic acid but gives minimal information about the identity and location of the mutation.
Other techniques employing ligase and polymerase extension assays are useful for determining whether a mutation is present at a defined location in an otherwise known target nucleic acid sequence. U.S. Pat. No. 4,988,617, for example, discloses a method for determining whether a mutation is present at a defined location in an otherwise known target nucleic acid sequence by assaying for the ligation of two natural oligonucleotides that are designed to hybridize adjacent to one another along the target sequence. U.S. Pat. No. 5,494,810 discloses a method that utilizes a thermostable ligase and the ligase chain reaction (LCR) to detect specific nucleotide substitutions, deletions, insertions and translocations within an otherwise known target nucleic acid sequence using only natural nucleic acids. U.S. Pat. No. 5,403,709 discloses a method for determining the nucleotide sequence by using another oligonucleotide as an extension and a third, bridging oligonucleotide to hold the first two together for ligation, and WO 97/35033 discloses methods for determining the identity of a nucleotide 3′ to a defined primer using a polymerase extension assay. Although the assays may be performed with a relatively high throughput, they are sequence specific and, thus require a different set of reagents for each target to be analyzed.
U.S. Pat. Nos. 5,521,065, 4,883,750 and 5,242,794 (Whiteley, et al.) disclose methods of testing for the presence or absence of a target sequence in a mixture of single-stranded nucleic acid fragments. The method involves reacting a mixture of single-stranded nucleic acid fragments with a first probe that is complementary to a first region of the target sequence and with a second probe that is complementary to a second region of the target sequence. The first and second target regions are contiguous with one another. Hybridization conditions are used in which the two probes become stably hybridized to their associated target regions. Following hybridization, any of the first and second probes hybridized to contiguous first and second target regions are ligated, and the sample is subsequently tested for the presence of expected probe ligation product.
Array Technology
Techniques employing hybridization to surface-bound DNA probe arrays are useful for analyzing the nucleotide sequence of target nucleic acids. These techniques rely upon the inherent ability of nucleic acids to form duplexes via hydrogen bonding according to Watson-Crick base-pairing rules. In theory, and to some extent in practice, hybridization to surface-bound DNA probe arrays can provide a relatively large amount of information in a single experiment. For example, array technology has identified single nucleotide polymorphisms within relatively long (1,000 residues or bases) sequences (Kozal, M., et al.,
Nature Med
. 7:753-759, July 1996). In addition, array technology is useful for some types of gene expression analysis, relying upon a comparative analysis of complex mixtures of MRNA target sequences (Lockart, D., et al., (1996)
Nat. Biotech
. 14, 1675-1680). Although array technologies offer the advantages of being reasonably sensitive and accurate when developed for specific applications and for specific sets of target sequences, they lack a generic implementation that can simultaneously be applied to multiple and/or different applications and targets. This is in large part due to the need for relatively long probe sequences, which are required to form and subsequently detect the probe/target duplexes. Moreover, this use of relatively long probes makes it difficult to interrogate single nucleotide differences due to the inherently small thermodynamic difference between the perfect complement and the single mismatch within the probe/target duplex. In addition, detection depends upon solution diffusion properties and hydrogen bonding between complementary target and probe sequences.
Mass Spectrometry Techniques
Mass spectrometry (MS) is a powerful tool for analyzing complex mixtures of compounds, including nucleic acids. In addition to accurately determining an intact mass, primary structure information can be obtained by several different MS strategies. The use of MS for DNA analysis has potential application to the detection of DNA modifications, DNA fragment mass determination, and DNA sequencing (see for example; Fields, G. B.,
Clinical Chemistry
43, 1108 (1997)). Both fast atom bombardment (
Myerson Joel
Sampas Nicholas M.
Sampson Jeffrey R.
Tsalenko Anna M.
Webb Peter G.
Agilent Technologie,s Inc.
Horlick Kenneth R.
Siew Jeffrey
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